BRPI0708733B1 - method for isolating 4-hydroxyproline rich polypeptides, using a composition based on them in the agronomic field and using them in the agronomic field ". - Google Patents

Abstract

The present invention relates to a method for the preparation of 4-hyroxy-L-proline or compositions with a high content of this aminoacid and the use thereof as agrochemical in the agronomical field and/or as a synthesis intermediate of chemical compounds.

Description

"METHOD FOR INSULATING 4-HYDROXYPROLINE-RICH POLYPEPTIDES, USING A COMPOSITION BASED ON THE SAME AGRONOMIC FIELD" AND THE USE OF A SAME AGRONOMIC FIELD -hydroxyproline and its uses in the agricultural field.

Especially, the present invention relates to a method for the preparation of 4-hydroxy-L-proline and compositions with a high content of this amino acid and its use as an agrochemical in the agricultural field and as a synthetic intermediate for chemical compounds. . 4-Hydroxyproline, a proline hydroxylate (4-HO-Pro) derivative, is an amino acid of considerable biological importance, which is typically found in a collagen. Hydroxyproline is currently obtained from protein sources of an animal, such as those of swine or bovine origin.

The methods for obtaining hydroxyproline rich oligopeptides provide that the source of animal protein is initially ground and stored in a low temperature environment until the time of treatment, which is composed of the following phases: - Acid hydrolysis (acidity is maintained throughout the operation to speed up the process and avoid any racemization). The temperature ranges from 70-100 ° C; - Filtration of insoluble products / decantation or solvent extraction of lipid or lipofluoric components; - Purification by ion exchange chromatography. The hydroxyproline content in the richest matrices can reach a maximum of 12 - 14% of the amino acid mass, with the presence of similar amounts of amino acids, eg proline (10 - 15%), which constitute the operating parameters for the critical separation; - Purification / color loss with active charcoal; - Ultrafiltration; - repeated crystallization; - Drying.

However, currently used processes have several disadvantages, such as obtaining a total free hydroxyproline or peptide concentration of less than 12 - 14% relative to the amino acid mass present, and the risk of contamination by bioactive peptides. dangerous. This may be associated with risks of contamination by a modified protein (with respect to the "non-pathological" form) called the "prion" responsible for the development of some serious pathologies, including bovine spongiform encephalopathy (BSE).

These risks recently led the European Union to ban the use of hydrolysed proteins of animal origin or derivation for use in organic farming.

Three groups of hydroxyproline-rich glycoproteins (hydroxyl-rich glycoprotein, HRGP) identified and characterized in plants are described in the literature (Showalter AM and Vamer JE (1988); Biochemistry of Plants, Vol.15, edited by Stumpf). PK & Conn, EE Academic Publisher, New York). These include: i) "arabinogalactan proteins", primarily located in the extracellular matrix and sometimes associated with the plasma membrane; ii) lecithins belonging to the Solanaceous family; and iii) extensins, the major components of the major cell walls where they covalently link the semicellulose matrix and pectins. They are especially abundant on the walls of cells growing in liquid media (Lamport, 1974; Cooper and Vamer, 1983; Qi et al., 1995).

Some of these extensins from carrot cell cultures have been broadly characterized in relation to their important role in controlling wall extension during cell growth (Lamport, 1969; Brysk and Chrispeels, 1972). More recently, new extensin-like proteins containing 20% proline and 20% hydroxyl proline have been extracted from soybean cell walls in a widely characterized culture (Averyhart-Fullard et al., 1988; Hong and Nagao, 1989 ). HRGP solubilization of cell walls was performed by various research groups with analogous methods. The first work is from 1969, when a first purification and identification of cell wall-bound proteins was performed (Lamport, 1969). A similar protocol based on the removal of glycoprotein-linked sugar chains from the wall with hydrochloric acid and consequent trypsin treatment was later applied to carrot cell cultures for the isolation and analysis of hydroxyproline-rich peptides derived from hydrolysis of the hydrochloric acid. wall glycoproteins (Cho and Chrispeels, 1976).

Protocols based on wall glycoprotein trypsinization have also been successfully applied to cotton cell cultures (QI et al., 1995).

Extensin-like proteins have been purified from soybean cell walls with processes based on strong trichloroacetic acid treatment, which hydrolyzes the glycoside bonds of these glycoproteins and also allows their separation from the cellulose matrix, and therefore isolated by HPLC techniques. more modern (Averyhart-Fullard et al., 1988). Analogous strong acid treatment and gel filtration processes were also used for the isolation and characterization of tomato cell culture wall extensins (Brownleader and Dey, 1993).

Only a few studies oriented towards the unique characterization of these glycoproteins and experimental verifications of their function in plant physiology are currently known. 4-HO-Pro (Plant Physiology (1984), "Effects of the Proline Analog L-Thiazolidine-4-carboxylic Acid on Proline Metabolism", vol. 74, pages 213 - 218, 1984) is also known to be capable of enhancing the proline level in plant tissues, reinforcing the plant's natural responses to alleviate / oppose the effects caused by abiotic stressors (eg extreme temperatures, drought, soil salinity, but also various types of chemical contamination) , and also to aid plant responses or the effect of exogenous phytiatric agents on phytopathogen control. 4-HO-Pro from animal sources is actually marketed for this purpose, but only in a mixture with other amino acids. For these applications, in fact it is available only in dilute form in hydrolyzed protein products, and especially hydrolysed products derived from chemical or enzymatic treatment of animal epithelium. This source, however, represents a possible risk of contamination due to the potential presence of bovine spongiform encephalopathy (BS A) vectors.

In the current state, the availability of alternative sources for obtaining 4-HO-Pro reducing the risks of biological contamination has become necessary.

It is an object of the present invention, therefore, to provide a method for obtaining 4-hydroxy-L-proline or compositions enriched with this amino acid in free form or in the form of oligopeptides, which have no risk of biological contamination.

Another object of the present invention is to provide 4-hydroxy-L-proline or compositions based on these amino acids which can be used as intermediates for the preparation of other chemicals.

Another object of the present invention is to present hydroxyproline based compositions which can be applied in the agronomic field and especially in organic farming.

In view of the above objects, according to a first aspect of the present invention, there is provided a method for preparing a 4-hydroxyproline compound composition according to claim 1.

Other accessory features of the method of the invention are set forth in the attached dependent claims 2-7. Applicant has found that by starting from plant cell cultures, and especially by properly treating the cell walls of various plant species, substantially free 4-hydroxy-L-proline rich amino acid compositions can be obtained. of other biological contaminants.

Especially, according to one embodiment of the invention, the 4-hydroxy-L-proline enriched compositions are obtained by fermentation of plant cell cultures, separation and lysis of cell cell walls.

According to another embodiment, the method of the invention also comprises protein degradation into peptides based on poly-4-HO-Pro released by chemically and / or enzymatically cell wall lysis, and optional partial or total purification. of the 4-HO-Pro thus obtained as free amino acid.

According to a preferred embodiment of the invention, the initial cell culture is based on tobacco BY2 cells.

The features and advantages of an embodiment of the method according to the present invention will become more apparent from the following illustrative and non-limiting description with reference to the following scheme which is composed of the following steps: a) fermentation of BY2 cells in a liquid culture ; B) separating BY2 cells from the fermentation medium;

C) homogenizing preferably with PBS containing 1% Triton X-100;

I d) cell wall precipitation and subsequent washing in PBS to remove cytoplasmic components; 1 e) boiling the cell walls in a 2 mM EDTA solution for pectin and starch removal; F) treating the walls with an acidic solution to separate the bound sugars in the wall proteins; G) digestion of the wall proteins in an acid and / or enzymatically medium; H) separation by chromatography of peptides and amino acids.

The a-h phases are conveniently carried out according to the following procedures and conditions: a) fermentation of the plant cells, preferably tobacco BY2, in a liquid medium; fermentation can be performed as described, for example, by Kato et al, 1972, by inoculating 50 ml of a dense cell culture into 1 liter culture medium containing 30 g / liter sucrose, 4.4 g / ml. Murashige / Skoog basal salt mixture (MS salt, Sigma), 0.1 g / liter Inositol, 1 mg / liter thiamine and 0.2 mg / liter 2.4 D and 0.18 g / liter of KH2PO4 at a pH of 5.7. Fermentation can also take place in different volumes and modified culture media compared to those proposed by Kato et al. b) BY2 cell separation: This can be accomplished by cell sedimentation and subsequent filtration of the liquid through a layer of Miracloth (Calbiochem) tissue, which retains the cells but allows the aqueous solution to pass through. According to another embodiment, it may be effected by centrifugation or also by simple decantation of the culture medium. The cells are then harvested with any collection system, for example a convenient spatula, and resuspended in PBS (1.44 g / liter NaH2PC> 4, 0.24 g / liter KH2PO4, NaCl 8 g / 1 at a pH of 7.4) and containing a detergent, for example 1% sodium dodecyl sulfate (SDS). The buffer suspension may differ from PBS in both chemical composition and pH. SDS may be substituted for other detergents that may be more suitable for the amount of cells used. c) BY2 cell homogenization: Cell homogenization can be carried out in a suitable container such as a ceramic mortar with a ceramic pestle. The amount of cells and relative volumes of buffer to be used varies with the previously chosen procedure. Homogenization can also be performed with mechanical or hydraulic presses or other commercially available apparatus suitable for frictional breaking of cells under cooled and uncooled conditions. d) cell wall precipitation: As soon as a homogeneous cell lysate has been obtained by homogenization, the cell sample is centrifuged, for example, at 3000 revolutions for approximately 15 minutes to precipitate the cell walls. Supernatant obtained from centrifugation is discarded, while the wall granules are washed three times with a buffer solution until all cytoplasmic components and detergents have been removed from the supernatant. e) Cell wall boiling: The cell walls thus obtained boil in the presence of a sequestering agent, eg 2mM EDTA, to eliminate pectins and starches that contaminate the wall preparation. The granules thus obtained are washed again in PBS. f) treatment of the walls with an acid solution: the cell wall granules are suspended in an acid solution, for example 0.1 N HCl (pH 1.0) and boiling, typically for about 1 hour. In this way all sugars covalently linked with the wall proteins are separated and placed in solution. Wall proteins are now accessible to attack by proteolytic enzymes that hydrolyze them into single peptides and / or amino acids. g) Wall protein digestion: This may be advantageously done by acidic or enzymatic means, or by using both methods, for example by altering enzymatic hydrolysis with acid hydrolysis. For acid treatment, inorganic acids, for example hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid are used; or organic acids such as acetic acid, trifluoroacetic acid, methanesulfonic acid, trifluoromethanesulfonic acid. When said digestion is performed enzymatically, proteolytic enzymes, such as trypsin (with methods typically described in Cho and Chrispeels, 1976) may be used, more preferably pepsin, which allows treatment to be done directly in the acid solution used for the hydrolysis of sugars, without the need for further washes or neutralizations with bases. h) Amino acid separation by chromatography: Further purification may be performed on the peptide / amino acid mixture extracted from the walls to separate the protein components having various molecular weights of sugars, mainly arabinoses. This can be done with weak ion exchange chromatography or gel filtration using Dowex50 columns (ion exchange) or Sephadex G10 or G15 gel filtration columns (gel filtration).

In another aspect, a polypeptide-based composition composed of 4-hydroxyproline obtained according to the method of the invention is disclosed.

According to another aspect, the use of 4-hydroxyproline from a polypeptide composition composed of 4-hydroxyproline, obtained according to the method of the invention, in the agronomic field, especially to give sapphires a greater tolerance to abiotic stresses, is presented. / or biotic, ie caused by phytopathogenic microorganisms. The applicant, in fact surprisingly, has found that 4-HO-Pro and the 4-HO-Pro containing compositions object of the present invention are capable of significantly inducing genes involved in plant self-defense, for example by induction of defense protein induced by pathogen attacks (Pathogen Related 1, PR1).

In particular, we have observed a significant effect of 4-hydroxy-L-proline and compositions enriched with this amino acid, object of the present invention, for controlling the development of biotic stress agents, such as phytopathogenic microorganisms, by inducing substances having an antifungal activity such as the defense protein PR1.

According to one embodiment, use is foreseen in the agronomic field, especially in biological agricultural crops of compositions composed of 4-HO-Pro in the presence of polysaccharides, typically arabinose chains, present in the walls of the BY2 tobacco cells, optionally separated from the protein matrix, but not removed from said composition.

In addition, the 4-HO-Pro obtained with the method of the invention also allows different associations to be obtained, for example with predetermined amino acids, or with polysaccharides or osmoprotectants of varying chemical and / or mechanical action, and also facilitates high purity 4-HO-Pro separation operations for synthetic uses (such as for the production of peptides with specific quaternary structures).

Another embodiment provides for the agronomic use of cell walls, preferably BY2 tobacco cells, by assimilating the degradation process described above to their natural metabolism produced on or within the plant, or in the soil, by the action of microorganisms or by the plant itself, and the synergistic effect that the hydroxyproline component and the arabinoside component produce to limit or alleviate the effects caused by abiotic and biotic stress. Hydroxyproline and / or compositions based on this amino acid obtained according to the method of the invention may be used for treating the entire plant or a portion thereof. They can therefore be applied to the leaves, but also to the trunk or stem, or roots by spraying solutions or suspensions or by incorporating granules or powders into the soil. The seeds may also be conveniently treated with the compounds object of the current patent as a result of the osmoprotective properties shown. Hydroxyproline and the compositions obtained according to the invention may be applied to all types of crops, monocotyledonous or dicotyledonous, and as a result, produce excellent assistance for the cultivation of, for example, cereals, fruit trees, vegetables, crops. " solanaceous ", various types of vegetables, but also ornamental plants, non-food oilseeds, lawns, etc.

Said compositions may be used alone or in a mixture with any other product traditionally used in agronomy, in any proportion the application procedure, both for crop protection (fungicides, herbicides, insecticides, nemoticides, biostimulants), but also for their protection. nutrition (fertilizers, various types of fertilizers, trace elements).

An object of the invention also relates to 4-HO-Pro based compositions derived from the process described above and of varying degrees of purity, optionally containing associated saccharides.

Application doses, expressed as a relationship to the weight content of 4-HO-Pro present in 1 liter or kg of composition of the invention, may range, for example, from 0.005 of 4-HO-Pro to 5,000 g of 4 -HO-Pro / hl, preferably from 0.05 g of 4-HO-Pro / hl to 500 g of 4-HO-Pro / hl, most preferably 0.5 g of 4-HO-Pro Pro / hl at 100 g of 4-HO-Pro / hl. This amino acid and the enriched compositions thereof have the additional advantage of being free from biological contaminants and, as a result, being able to be freely used in the field of organic farming.

According to another aspect of the invention, the use of 4-HO-Pro or 4-hydroxyproline-containing polypeptides as intermediates for the preparation of synthesis molecules is envisaged.

For illustration but not for purposes of limitation of the present invention, some examples are given below concerning the preparation of 4-HO-Pro enriched compositions according to the invention and the biological activity thereof. EXAMPLE 1 Isolation of hydroxyproline rich peptides from BY2 cell walls.

BY2 cells were obtained by fermentation performed as described by Kato et al., 1972. 50 ml of a dense cell culture was used as the starting material for the inoculation of 1 liter culture medium containing 30 g / liter. of sucrose, 4.4 g / liter Murashige / Skoog basal salt mixture (MS salt, Sigma), 0.1 g / liter Inositol, 1 mg / liter thiamine, 0.2 mg / liter 2.4 D, 0.18 g / liter of KH2PO4 at a pH of 5.7. The culture is then incubated at 27 ° C in the dark under constant orbital shaking (110 rpm) and after 8 days the cells are filtered through a layer of Miracloth tissue (Calbiochem), and homogenized in a mortar in the presence of SDS ( sodium dodecyl sulfate) 1% (or Triton X-100, 1%) in a PBS buffer solution (phosphate buffered saline: 1.44 g / liter NaH2PO4, 0.24 g / liter KH2PO4, NaCl 8 g / liter at a pH of 7.4). The resulting lysate is first boiled in the presence of EDTA, treated with 0.1 N HCl and then digested with pepsin in an acidic environment. This procedure leads to the solubilization of the protein fraction present in the cell walls, quantifiable with the Bradford test (Bradford, 1976) or with the addition of ninhhydrin.

More specifically: BY2 cells (300 g) derived from 1 liter culture are homogenized in a mortar containing about 300 ml PBS containing 1% SDS (Triton X-100). The obtained lysate is centrifuged at 3000 rpm to precipitate the cell walls which are then washed with additional amounts of PBS for removal of cytoplasmic components. The product obtained, defined as composition number 1, can be used for agronomic applications. Additional boiling of the cell walls in 150 ml of 2mM EDTA allows the removal of pectins and starch. The walls are then treated with 0.1N HCl (150 ml) to separate the bound proteins from the wall proteins to give composition number 2 which, as soon as it is neutralized with NaOH and properly diluted, can be used for agronomic applications. .

Pepsin (1 mg / ml) is then added for digestion of the wall proteins, keeping the pH at a value of 1.0 in a volume of 150 ml. The amount of protein obtained from digestion is measured with the Bradford method using BSA (bovine serum albumin) as a standard.

The peptides thus obtained (about 120 - 130 mg) in acid solution which also contain the saccharide fraction are lyophilized and resuspended in a buffer solution (pH 6) for the biological test (composition No. 3). EXAMPLE 2 Further Purification of Hydroxyproline Rich Peptides from BY2 Cell Walls: Chromatographic separation of amino acids. Composition No. 3, derived from acid and pepsin wall digestion, was lyophilized and resuspended in 10 - 15 ml of a 10 mM pyridine formate buffer solution, pH 3.3, to be applied to a chromatographic column. previously washed and equilibrated with 1mM pyridine buffer solution.

Columns containing a Dowex 50WX2-400 (Sigma) resin, used in weak ion exchange chromatography, are loaded with material derived from wall acid and protein digestions. The column thus charged is first eluted with 1mM pyridine formate, pH 3.3, and then with a 750mM pyridine buffer solution. Charged molecules, such as amino acids and peptides, are removed by the column and are not eluted with the first pyridine formate elution. The 1mM buffer solution elutes only neutral or weakly charged molecules such as sugars. Elution with a higher pyridine buffer solution also allows charged molecules such as amino acids and peptides to be eluted.

In gel filtration chromatography, columns containing Sephadex G10 (Sigma) resin allow molecules with a molecular weight of less than 700 Da to be separated. Gel filtration explores the size of the molecules for separation, Larger molecules, such as protein fragments and larger peptides, are first eluted, unlike small molecules, such as amino acids or simple sugars, which are retained by resin porosity, and are eluted with larger volumes of buffer solution. The same pyridine formate buffer solution at a pH of 3.3 can also be applied to the Sephadex columns to elute the peptides and later the amino acids alone derived from cell wall protein hydrolysis.

In either case, the advantage of using a pyridine formate buffer solution is its high volatility, which allows it to be easily eliminated after lyophilization to recover the peptide component free of contamination by salts or other organic molecules. . EXAMPLE 3 Expression of the abiotic and biotic anti-stress genes PDH, GSTF7, PR1 after treatment of plants with hydroxyproline-containing compositions. Composition number 3, derived from acid and pepsin digestion of the walls, was lyophilized and resuspended in 15 ml of a 250 mM NaOH solution to have a final pH of 6.0. 1 ml of this solution was further diluted in 10 ml of distilled water to be used directly on Arabidopsis thaliana plants. The Arabidopsis Columbia ecotype plants, grown in soil for 3-4 weeks and exposed to light over a period of 16h, were sprayed onto the leaf surface with composition number 3. After 24h, some of the leaves were removed from each sample and Total RNA was extracted with the Sigma kit. CDNA was synthesized using Promega and Fermentas products.

Quantitative PCR experiments were performed on various genes involved in plant defense response using a kit purchased from Ambion, in which two primer / competitor pairs are used for amplification of the 18S ribosomal RNA gene, which represents the standard of control. Gene expression analysis was performed on three genes, which represent excellent molecular markers to reveal and quantify the plant's defensive response to both abiotic and biotic stress. The genes analyzed are those encoding the enzyme proline dehydrogenase (PDH), described by Hua et al., 2001, that of glutathione s-transferase (GST-F7) (Bechtold et al, 1993), and that of protein PR1 (protein 1 related to pathogenesis) (Lebel et al., 1998). The first two genes are involved in the plant defense response to stress the abiotic type, which is usually induced by an increase in plant proline. The latter gene, on the other hand, is involved in the biotic stress response, i.e. it is activated after the attack of pathogens and induces a response generally mediated by salicylic acid.

In several experiments, it is demonstrated how treatment with composition number 3 leads to significant induction of both the PDH and GST-F7 genes (abiotic stress response) and also to the PR1 gene (biotic stress response). EXAMPLE 4 Synergy of the presence of sugars in the expression of genes responsible for abiotic and biotic stress relief.

Induction experiments of the same gene were performed using composition material number 3 fractionated on a Dowex50 column. Different fractions, obtained by elution with a 1mM pyridine formate buffer containing mainly sugars, were tested on plants under the same conditions as described above and produced a weak inductive effect. The protein components, both the larger peptide fractions and the amino acids alone (mainly represented by hydroxyproline) produced a significantly higher response in stress marker gene expression. The effect on gene expression produced by composition number 3 is greater than that produced by fractional components alone, suggesting a synergistic action of the glycoside component along with the protein component. The effect of plant stress response was also evaluated by analyzing other genes, important markers of both abiotic and biotic stress. The other genes analyzed, which produced analogous results, are SAG29 (Senescence Associated Gene 29), SAG12 (Senescence Associated Gene 12), WHY1 (Whirly 1), TGA2 (transcription factor), COR47 (involved in saline stress and drought). , CAT3 (coding for a catalase), LOX2 and ERF1 (both involved in biotic stress response). EXAMPLE 5 Relief of the effects of abiotic stress on bean plants.

Bean plants grown in a high salinity medium were treated with composition number 3 by applying a dose of 50 g / hl of amino acid-protein component per plant (thesis 2). These plants were compared with plants grown in the same saline but not treated (thesis 3) with plants grown in a medium of the same composition but not saline (thesis 1). 4 plants were used for each thesis, kept in a greenhouse for 8 days and then visually compared. Untreated thesis 3 had extensive tissue necrosis, while thesis 2 had a high reduction in the effects of high salinity, having a similar development and condition to the plant grown in the absence of salinity and demonstrating the ability of the composition. # 3 relief from the effects of typical abiotic stress. The same experiment was obtained with compositions number 1 and 2, again with a dose of 50 g / hl of amino acid-protein component, giving similar results.

Claims (7)

Method for the isolation of 4-hydroxyproline rich polypeptides, characterized in that it comprises the following steps: a) fermentation of a tobacco cell culture; b) separating the tobacco cells from the fermentation medium; c) homogenization of tobacco cells; d) boiling cell walls to remove pectins and starches; e) treating the cell walls with an acidic solution to separate the bound sugars in the wall proteins; (f) digestion of wall proteins by pepsin hydrolysis, Method according to Claim 1, characterized in that it comprises, between steps c) and d), a step of precipitation of the cell walls and washing for the removal of the cellular eitoplasmic components. Method according to either of Claims 1 or 2, characterized in that the digestion of the wall proteins of step f) is carried out by alternating acid hydrolysis and acid hydrolysis. Method according to any one of claims 1, 2 or 3, characterized in that it comprises an additional separation step g) by chromatography of the peptides and amino acids. Method according to any one of claims 1, 2, 3 or 4, characterized in that the cell culture is a BY2 tobacco cell culture. Use of a composition based on 4-hydroxyproline rich polypeptides obtained according to the method defined in any one of claims 1 to 5 in the agronomic field characterized in that the crops have a greater tolerance to abiotic stress and / or biotic. Use of 4-hydroxyproline rich polypeptides obtained according to the method defined in any one of claims 1 to 5 in the agronomic field, characterized in that it is to provide crops with greater tolerance to abiotic and / or biotic stress.